DE69728377T2 - IMPROVED MUTANT OF (2.5 DKG) REDUCTASE A - Google Patents

Abstract

Mutants of 2,5-diketo-D-gluconic acid reductase A an enzyme used to produce 2-keto-L-gluconic acid, a precursor of ascorbic acid (vitamin C), are prepared by site-directed mutagenesis. These mutants may exhibit one or more of the following characteristics: improved temperature stability, increased resistance to substrate inhibition, increased turnover of the substrate by the enzyme and increased affinity for the substrate.

Description

The The present invention relates to improved mutated forms an industrially valuable enzyme. In particular, the Invention to mutated forms of 2,5-diketo-D-gluconic acid (2,5-DKG) reductase A, Naturally occurring variants of 2,5-DKG reductase. The mutated forms show an improved catalytic activity to 2,5-DKG stereoselective in 2-keto-L-gluconic acid (2-KLG), a precursor of ascorbic acid (Vitamin C) to metabolize. The mutated forms may include one or more of the exercise the following properties: improved temperature stability, Increased resistance to substrate inhibition, increased conversion of the substrate by the enzyme and increased affinity for the substrate.

Because of a widening health awareness of people everywhere There was an increased need for vitamin C in the world. To the need for ascorbic acid also contributes its widespread use as an antioxidant for preservation of foods. An approach to satisfying this demand is an elevated one To achieve production of 2-KLG, an intermediate in the production of ascorbic acid. The intermediate 2-KLG can easily be replaced by an acid or hare-catalyzed cyclization can be converted to ascorbic acid. It also has a higher one stability and storage stability as ascorbic acid. That's why it's more practical, instead of directly producing ascorbic acid, 2-KLG for the subsequent conversion to ascorbic acid storage.

A Series of species of a first group of microorganisms, Erwinia, Acetobacter and Gluconobacter Generate 2.5 DKG from D-glucose. A second group of microorganisms from the coryneform group of bacteria (Corynebacterium, Brevibacterium and Arthrobacter), as well as species of Micrococcus, Staphylococcus, Pseudomonas, Bacillus and Citrobacter, are capable of 2,5-DKG, produced by a microorganism of the first group, to 2-KLG convert. A tandem fermentation or cofermentation more appropriate Microorganisms to produce 2-KLG have been simplified by the combination of the relevant properties of both the Erwinia sp. as well as that of Corynebacterium sp. in a single microorganism (Anderson et al., Science, 23: 144-149 (1985)). This was accomplished by the identification of 2,5-DKG reductase in Corynebacterium sp., which converts 2,5-DKG to 2-KLG. The Gene for this reductase was then cloned into Erwinia herbicola, a Bacterium of the family Enterobacteriaceae, which is D-glucose converted into 2.5-DKG in a single fermentation. The resulting recombinant bacterial strain, with 2,5-DKG reductase As the central enzyme, D-glucose was able to undergo 2-KLG in a single fermentation process (Lazarus et al., Fourth ASM Conf. Genet. Molec. Biol. Indust. Microorg., 187-193 (1989)).

The Improvement of the catalytic activity of 2,5-DKG reductase in the single fermentation process is a significant way to to increase the production of 2-KLG. Also could be a purified 2,5-DKG reductase A with elevated catalytic activity in an in vitro process for the Conversion of 2.5-DKG to 2-KLG can be used. For example, such would be a process the continuous production of 2-KLG by immobilization allow the purified enzyme on a solid support material.

kann die Effizienz einer enzymatischen Reaktion durch zwei kinetische Parameter, kcat und Km, gemessen werden. Die Katalysegeschwindigkeitskonstante, kcat, auch bekannt als die Umsetzungszahl, ist ein Maß für das Zusammenbrechen des Enzymsubstrat-(ES)-Komplexes. Es steht auch für die maximale Anzahl an Substratmolekülen (S), die zum Produkt (P) über einen ES-Komplex pro aktives Zentrum des Enzyms (E) pro Zeiteinheit umgewandelt werden. Vmax ist die Maximalgeschwindigkeit oder -rate der enzymkatalysierten Reaktion, wenn das Enzym mit Substrat gesättigt ist. Deswegen ist Vmax bei gesättigter Substratkonzentration konstant und bleibt mit jedem Anstieg in der Substratkonzentration unverändert. Bei gesättigten Substratkonzentrationen steht kcat in Beziehung mit Vmax und der Gesamtenzymkonzentration, [E_T], durch die folgende Gleichung: Vmax = kcat [E_T]. Die Michaelis-Konstante, Km, ist die Substratkonzentration, bei der die Geschwindigkeit gleich Vmax/2 ist. Folglich ist Km ein Maß für die Stärke des ES-Komplexes. In einem Vergleich von Km-Werten repräsentiert ein niedrigerer Km einen Komplex mit einer stärkeren, günstigeren Bindung, wohingegen ein höherer Km einen Komplex mit einer schwächeren, weniger günstigen Bindung repräsentiert. Das Verhältnis kcat/Km, als Spezifizitätskonstante bezeichnet, repräsentiert die Spezifizität eines Enzyms für ein Substrat, das heißt die katalytische Effizienz pro Enzymmolekül für ein Substrat. Je größer die Spezifizitätskonstante ist desto bevorzugter ist das Substrat durch das Enzym.According to the Michaelis-Menten scheme outlined below

For example, the efficiency of an enzymatic reaction can be measured by two kinetic parameters, kcat and Km. The catalytic rate constant, kcat, also known as the conversion number, is a measure of the breakdown of the enzyme substrate (ES) complex. It also represents the maximum number of substrate molecules (S) that are converted to product (P) via an ES complex per active site of the enzyme (E) per unit time. Vmax is the maximum rate or rate of the enzyme-catalyzed reaction when the enzyme is saturated with substrate. Therefore, Vmax is constant at saturated substrate concentration and remains unchanged with each increase in substrate concentration. For saturated substrate concentrations, kcat is related to Vmax and the total enzyme concentration, [E _T ], by the following equation: Vmax = kcat [E _T ]. The Michaelis constant, Km, is the substrate concentration at which the velocity is equal to Vmax / 2. Consequently, Km is a measure of the strength of the ES complex. In a comparison of Km values, a lower Km represents a complex with a stronger, more favorable bond, whereas a higher Km represents a complex with a weaker, less favorable bond. The ratio kcat / Km, called the specificity constant, represents the specificity of an enzyme for a substrate, that is, the catalytic efficiency per enzyme molecule for a substrate. The larger the specificity constant, the more preferred the substrate is by the enzyme.

impressive Levels of 2-KLG were measured with a 2,5-DKG reductase from Corynebacterium (2,5-DKG reductase A, also known as 2,5-DKG reductase II) (Anderson et al., Science, 230: 144-149 (1985); Miller et al., J. Biol. Chem., 262: 9016-9020 (1987)), expressed in suitable host strains (2.5 DKG manufacturer) such as B. Erwinia sp. Nevertheless, these results were achieved that of 2,5-DKG reductase A from a low specificity constant for 2.5 DKG was reported.

These low reported specificity constant for 2,5-DKG reductase A is in contradiction to a second, homologous Corynebacterium-2,5-DKG reductase (2,5-DKG reductase B, also known as 2,5-DKG reductase I), from the one higher specificity constant for 2.5 DKG (Sonoyama and Kobayashi, J. Ferment, Technol., 65: 311-317 (1987)). additionally both 2,5-DKG reductases are homologous to a number of known aldose and keto reductases, the higher ones Spezifizitätskonstanten for her known ones Have substrates. Because Corynebacterium does not naturally encounter 2.5-DKG, it is not surprising that this compound is a poor substrate for 2,5-DKG reductase A. Such findings show suggest that the active site of 2,5-DKG reductase A is not optimal for the catalytic conversion of 2,5-DKG to 2-KLG is configured. therefore It seems that in order to reduce the specific activity of 2,5-DKG reductase A in to optimize the single fermentation process, amino acid substitutions by site-directed mutagenesis at the active site of the enzyme placed Need to become.

In addition to Improving the kinetic parameters of an enzyme can be site-directed Mutagenesis the structural stability by amino acid substitutions, increase deletions or insertions. The following are examples structurally stabilizing mutations. Introducing new ones Disulfide bonds to covalent cross-links between different Creating a protein was used to create the thermal stability of lysozyme of bacteriophage T4 (Matsumura et al., Nature, 342: 291-293 (1989)), of the bacteriophage λ repressor (Sauer et al., Biochem., 25: 5992-5998 (1986)), E. coli dihydrofolate reductase (Villafranca et al., Biochem., 26: 2182-2189 (1987)) and subtilisin BPN '(Pantoliano et al., Biochem. 26: 2077-2082 (1987)). There is a computer program (Pabo et al Biochem., 25: 5987-5991 (1986)), which determined an effective search of the crystallographically Three-dimensional structure of a protein allowed to those sites suggest where to insert from two cysteines to disulfide bridges to lead could. Such bonds would the conformation in big scale do not bother, while she the local Stabilize conformation.

The amino acid substitutions of alanine for glycine in the α-helix have been shown to enhance the thermal stability of the bacteriophage λ repressor (Hecht et al., Struct. Funct. Genet., 1: 43-46 (1986)) and the neutral protease from Bacillus stearothermophilus (Imanaka et al., Nature, 324: 695-697 (1986)). An increase in the melting temperature, T _m , for the bacteriophage T4 lysozyme was accomplished by two amino acid substitutions of proline for alanine and alanine for glycine (Matthews et al., Proc. Natl. Acad Sci., USA, 84: 6663-6667 ( 1987)). From the replacement of amino acids in the hydrophobic core of a protein by aromatic radicals, such. Tyrosine, particularly at positions close to already existing aromatic side-chain clusters, has been shown to increase the thermal stability in kanamycin nucleotidyltransferase (Liao et al., Biochem., 83: 576-580 (1986)) and in U.S. Pat the bacteriophage λ repressor (Hecht et al., Biochem., 81: 5685-5689 (1984)).

Transcriptional and translational control sequences in expression vectors are key elements required for the production of proteins in bacteria to a high degree. The E. coli Trp, bacteriophage λP _L , E. coli lac UV5 and Trp-lacUV5 fusion (tac) promoters are among the most commonly used prokaryotic promoters (de Boer et al., Proc. Natl Acad Sci., USA, 80: 21-25 (1983); Sambrook et al., Molecular Cloning, Cold Spring Harbor Press (1989); Remaut et al., Gene, 15: 81-93 (1981)). The translation efficiency of the message, mRNA stability and intrinsic protein stability are major factors of high expression.

Site-directed Mutagenesis using synthetic DNA oligonucleotides with the desired Sequence allows the substitution, deletion or insertion of selected nucleotides within a DNA sequence that encodes a protein of interest. Recombinant DNA procedures are used to obtain the desired mutation by substitution of the synthetic sequence for the target sequence. The Development of plasmids containing an origin of replication, derived from a filamentous Bacteriophages (Vieira and Messing, Methods in Enzymology, 153: 3-11 (1987)) allows the cloning of fragments in single-stranded forms of plasmids, capable to autonomous replication. The use of such plasmids eliminated the tedious business of subcloning DNA fragments of plasmids into filamentous bacteriophage vectors. Kits to perform of site-directed mutagenesis are commercially available.

mutants 2,5-DKG reductase A with properties similar to those of native Enzyme would vary useful. Especially one or more of the following properties: improved Temperature stability, increased resistance against substrate inhibition, increased conversion of the substrate by the enzyme and increased affinity for the substrate, would be useful for commercial usefulness of the enzyme.

Unfortunately it is impossible, if proteins are not areas of essential sequence or structural Share homology, generalize between proteins, based on a cheap Mutation of a protein, precise predict where the sequence encoding another protein changed should be to improve the appearance of that protein. Therefore it is necessary to have an analysis of the precise structural and functional Characteristics of the particular one to be changed Protein. This suggests which amino acids to change a desired one Produce result, such. B. increased catalytic efficiency or thermal stability.

Increasingly is the correlation between the structures of known proteins and the sequence of a target protein using computer simulations (V.F. van Gunsteren, Prot. Engin., 2: 5-13 (1988); M.M. Yang et al. in: Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle, Ed.), Springer-Verlag, Germany (1990), pp. 209-218), databases (Moult, J., et al., Proteins, 1: 146-163 (1987); P. Klein et al., Biopolymers, Vol. 25: 1659-1672 (1986); H. Nakashima et al., J. Biochem., 99: 153-162 (1986); G. Deleage et al., Prot. Engin., 1: 289-294 (1987)); neural networks Qian et al., J. Molec. Biol., 202: 865-884 (1988); L.H. Holley et al al., Proc. Natl. Acad. Sci. (USA), 86: 152-156 (1989); H. Bohr et al. FEBS Lett., 241: 223-228 (1988)); or expert systems (B. Robson et al., J. Molec. Graphics, 5: 8-17 (1987)) made. See, in general, G.R. Fasman, TIBS, 14: 295-299 (1989)).

The Use of computers or computational methods in the analysis the structure of proteins is z. In U.S. Patents 4,704,692 (Ladner); 4,760,025 (Estell et al.); 4,853,871 (Pantoliano et al.) and 4,908,773 (Pantoliano et al.).

Sequence comparisons, carried out using the sequence of 2,5-DKG reductase, showed that it is a member of a larger superfamily monomeric, NADPH-dependent prokaryotic and eukaryotic carbonyl reductases as the aldo-keto reductases (Career et al., Exp. Eye Res., 49: 377-388 (1989); Bohren et al., J. Biol. Chem., 264: 9547-9551 (1989)). Members of this Close enzyme family a: biosynthetic enzymes such. Bovine prostaglandin F synthase; detoxifying enzymes such. B. chlorodeconuctase and aflatoxin b1 reductase; and structural proteins without identified enzymatic activity such. B. rho-crystalline from frog lentils.

The human aldose reductase enzyme was characterized and expanded examined. Aldose reductase has been associated with diabetic complications; it is believed by her to be the reductionist from glucose to sorbitol in diabetic patients causing what in diabetic cataracts and neuropathology with long-term diabetes connected results. Significant efforts have been made to find specific aldose reductase inhibitors to diabetic To prevent complications in humans (Frank, Opthamology, 98: 586-593 (1991); Zenon et al., Clinical Pharmacy, 9: 446-456 (1990)). Because of their possible importance In human health, the crystal structure of the human Aldose reductase is resolved by several groups, either as the harvest enzyme (Wilson et al., Science, 257: 81-84 (1992), in complex with NADPH cofactor or the cofactor analogue ATP-ribose (Rondeau et al., Nature, 355: 469-472 (1992); Borhani et al., J. Biol. Chem., 267: 24841-24847 (1992) or in complex with the inhibitor zopolrestat (Wilson et al., Proc. Natl. Acad. Sci., 90: 9847-9851 (1993)). Recently became the structure of another member of the aldo-keto-reductase family, alpha HSD, also solved (Hoog et al., Proc. Natl. Acad. Sci., 91: 2517-2521 (1994). These structures show that the aldo-keto reductases are eightfold alpha / beta-parallel Tons are, also known as the "TIM-tons" motif, after the triosephosphate isomerase, where it was first described. This is an extremely common protein folding with about 17 known examples; approximately 10% of all enzymes whose structures are known are "TIM tons" (Farber and Petsko, TIBS, 1990: 228-235 (1990).

The structure of human aldose reductase shows a number of features. The aldose reductase α / β barrel is composed of eight beta strands that form the "core" of the barrel, surrounded by eight alpha helices connected by loops of varying lengths to the beta strands. As in all known TIM-tons enzymes, the loops found at the C-terminal ends of the beta strands comprise the active site of the enzyme, where substrate and cofactor bind and catalysis occurs. NADPH is bound at the top of the barrel in an extended conformation, with the nicotinamide ring from which the hydride transfer takes place occupies approximately the exact center of the ton. The orientation of the cofactor nicotinamide ring is what would be expected for a class A reductase in which the pro-R hydrogen enters the substrate-binding pockets. There are two additional secondary structural features on the aldose reductase ketone: two additional alpha helices (referred to as H1 and H2) found on the loops of the amino acids connecting the beta strand seven and the alpha helix seven, and at C -terminal "tail" after the alpha-helix eight. The structure of aldose reductase indicates that this C-terminal tail protrudes beyond the top of the barrel to form part of the active site.

The WO94 / 05772 provides mutants that have specific modifications contain the 2,5-DKG reductase A, which increased stability, expression and / or catalytic activity exercise could and which could have different Km and Vmax.

The The present invention makes mutant forms more enzymatically active prokaryotic 2,5-DKG reductase A ready.

SUMMARY THE INVENTION

The The present invention provides mutants that have specific modifications the 2,5-DKG reductase A, and materials and methods that are useful in manufacturing these proteins, as well as modified microorganisms and cell lines, the useful are in their production. In particular, the invention provides a mutant form of 2,5-DKG reductase A ready, with an improved Ability, 2,5-DKG into 2-KLG and with an amino acid substitution in position 22, as defined in SEQ ID NO: 1. Other Aspects of Close invention Expression constructs and products thereof for the modified 2,5-DKG reductases, as well as cloning vectors containing the DNA which encode the modified 2,5-DKG reductases. Especially the invention provides a DNA construct with a structural gene which contains at least one mutated codon, said gene being a mutant encoded the invention. Also provided is a host cell which is transformed with an expression vector which is a DNA construct of the invention includes.

The DNA encoding wild-type 2,5-DKG reductase A is modified using site-directed mutagenesis, wherein a single-stranded form the gene used is the generation of a change at a selected Within the coding region of the 2,5-DKG reductase A allows. By This procedure will be a change introduced in isolated DNA, the Encoded 2,5-DKG reductase A, which after expression of the DNA in the substitution of at least one amino acid at a predetermined one Site in the 2,5-DKG reductase A results.

The modified 2,5-DKG reductases and coding sequences of the invention can Have one or more of the following properties: Improved Temperature stability, increased Resistance to substrate inhibition, increased conversion of the substrate through the enzyme and increased affinity for the Substrate. The modified 2,5-DKG reductases can have different Km and Vmax values to have.

SHORT DESCRIPTION THE DRAWINGS

1 shows an expression vector for the 2,5-DKG reductase A gene;

2 shows an expression vector for the production of mutant forms of 2,5-DKG reductase A; and

3 shows the plasmids ptrpl-35.A and prtpl-35.B.

4 schematically shows an algorithmic model for 2,5-DKG reductase A (SEQ ID NO: 1).

5 Figure 3 shows a comparison of the predicted secondary elements in 2,5-DKG reductase A based on the algorithm and homology model.

6 Figure 4 shows a protein sequence assembly of 2,5-DKG reductase A and B with human aldose reductase. The boxes represent secondary structural elements in aldose reductase (SEQ ID NO: 2) (SEQ ID NO: 3).

7 shows the substrate kinetics of the 2,5-DKG reductase A mutant F22Y and the 2,5-DKG reductase B mutant Y23F in comparison with the wild-type 2,5 DKG reductases A and B.

8th shows the substrate kinetics of the 2,5-DKG reductase B mutant N50A in comparison with the wild-type 2,5 DKG reductases A and B.

9 shows the substrate kinetics of the 2,5-DKG reductase A mutant A272G in comparison with the wild type 2,5 DKG reductases A and B.

10 shows the substrate kinetics of the 2,5-DKG reductase A mutant F22Y / Q192R, the 2,5-DKG reductase A mutant F22Y / A272G and the 2,5-DKG reductase A mutant Q192R / A272G in comparison with the wild-type 2,5-DKG reductases A and B.

11 shows the cofactor Km of the 2,5-DKG reductase A mutants F22Y, Q192R, A272G and F22Y / A272G in comparison with the 2,5-DKG reductases A and B.

12 shows the analysis of the thermal denaturation of selected mutants in comparison with the wild-type 2,5-DKG reductases A and B.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As As used herein, the term "wild-type" 2,5-DKG reductase A refers to a protein, which is capable of stereoselective conversion of 2,5-DKG Catalyze 2-KLG. The wild-type enzyme is the enzyme that won is from Corynebacterium sp., obtained from ATCC strain No. 31090, as described in U.S. Patent No. 5,008,193.

Of the Term "wild-type" 2,5-DKG reductase B refers to a protein that is capable of being stereoselective to catalyze the conversion of 2,5-DKG to 2-KLG. The wild-type enzyme is the enzyme obtained from Corynebacterium sp. shs752001 as described by Hardy et al. in U.S. Patent 4,945,052.

As As used herein, the term "mutant" refers to a protein such as z. "Wild-type" 2,5-DKG reductase A or "wild-type" 2,5-DKG reductase B, to a protein with a related amino acid sequence. It contains, however one or more amino acid substitutions, Deletions or insertions of amino acid residues. These radicals were selected using certain approaches. An approach contains the use of secondary structural Predictions to 2,5-DKG reductase A of an eight-stranded α / β barrel structure assigned. A number of modifications can be made to to modify the gene to encode mutants of the enzyme with improved properties compared to the wild-type enzyme, and by 2.5 DKG stereoselectively in 2-KLG convert.

It is well understood in the art that many of the compounds, those in the current description be discussed, such. As proteins and the acidic derivatives of Sugars, in a series of ionization states may exist in dependence from their surrounding media, if they are in solution or outside the solutions, from which they were prepared if they were in solid form. at the use of a term such as gluconic acid such molecules It is intended to designate all ionization states of the organic molecule, which he refers to. Consequently, refer z. B. both "D-gluconic acid" and "D-gluconate" on the same organic Unit and are not designed to determine particular ionization conditions. It is well known that D-gluconic acid exist in unionized form can or z. As the sodium, potassium or other salt. The ionized or unionized form in which the compound for the Disclosure is either out of context for one of ordinary skill in the art be obvious or meaningless. Thus, the 2,5-DKG reductase A protein and their different mutants in a series of ionization states in dependence of the pH exist. All these ionization states are encompassed by the terms "2,5-DKG reductase A "and" mutated form of 2,5-DKG Reductase A ".

The term "expression vector" includes vectors capable of expressing DNA sequences contained therein, where such sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not expressly stated, that expression vectors must be replicable in the host organisms either as episomes or as an integral part of chromosomal DNA. It is clear that a lack of replication would actually make them inoperable. As a result, "expression vector" is also given a functional definition. Generally Expressi vectors useful in recombinant DNA techniques, often in the form of "plasmids". Plasmids refer to either circular double-stranded DNA molecules or circular single-stranded DNA molecules that contain an origin of replication. These DNA molecules, in their vector form, are not coupled to the chromosomes. Other effective vectors commonly used are phage and circular DNA. In the present specification, "plasmid" and "vector" are often used interchangeably. However, the invention is intended to encompass other such forms of expression vectors which serve equivalent functions and which are known or hereafter known.

From the term "construct" is intended that he broad plasmids, vectors, etc. and fragments thereof (such. Cassettes and gene sequences).

"Recombinant host cells", "host cell", "cells", "cell cultures", etc., become interchangeable used to identify individual cells, cell lines, and cell cultures to identify harvested cells that are infected with the recombinant vectors of the invention have been transformed or are intended to to transform it with it. The terms also include the precursors of Cells that originally got the vector.

"Transformation" refers to every procedure to change the DNS content of the host.

This includes in vitro transformation processes such as By calcium chloride, calcium phosphate or DEAE-dextran mediated transfection, conjugation, electroporation, nuclear injection, Phage infection or such other means to be controlled To effect DNA uptake, as known in the art.

The Terms "amino acid" and "amino acids" refer to all Naturally occurring L-α-amino acids. From This definition is thought to be only leucine, ornithine and Includes homocysteine. The amino acids are identified by either their single-letter or three-letter designations:

• I. Geladene Aminosäuren saure Reste: Asparaginsäure, Glutaminsäure basische Reste: Lysin, Arginin, Histidin
• II. Ungeladene Aminosäuren hydrophile Reste: Serin, Threonin, Asparagin, Glutamin aliphatische Reste: Glycin, Alanin, Valin, Leucin, Isoleucin apolare Reste: Cystein, Methionin, Prolin aromatische Reste: Phenylalanin, Tyrosin, Tryptophan

Tabelle 1 Ausgangsrest konservative Substitutionen Ala ser Arg lys Asn gln; his Asp glu Cys ser, ala Gln asn Glu asp Gly pro His asn; gln Ile leu; val Leu ile; val Lys arg gln; glu Met leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu These amino acids can be classified according to the chemical composition and the properties of their side chains. They are roughly classified into two groups, charged and uncharged. Each of these groups is divided into subgroups to more accurately classify the amino acids:

• I. charged amino acids acid residues: aspartic acid, glutamic acid basic residues: lysine, arginine, histidine
• II. Uncharged amino acids hydrophilic residues: serine, threonine, asparagine, glutamine aliphatic residues: glycine, alanine, valine, leucine, isoleucine apolar residues: cysteine, methionine, proline aromatic residues: phenylalanine, tyrosine, tryptophan

Table 1 output rest conservative substitutions Ala ser bad lys Asn gln; his Asp glu Cys water, ala Gln asn Glu asp Gly Per His asn; gln Ile leu; val Leu ile; val Lys bad luck; glu mead leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Significant changes in function or stabilization are selected by selecting Substitutions produced that are less conservative than those in Table 1, that is by selecting of residues that are more significant in their effect on maintenance of (a) the structure of the polypeptide backbone in the region of substitution, such as B. as a leaf or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the mass of the side chain. The substitutions that are generally expected to be they are the strongest changes will be those in which (a) a hydrophilic group, e.g. As serine or threonine, is substituted for (or by) a hydrophobic radical, z. Leucine, isoleucine, phenylalanine, valine or alanine; (b) a Cysteine or proline is substituted for (or by) any other rest; (c) a moiety having an electropositive side chain, z. As lysine, arginine or histidine, is substituted for (or by) an electronegative radical, e.g. Glutamic acid or aspartic acid; or (d) a remainder with a large one Side chain, z. As phenylalanine, is replaced by (or by) one, the no Side chain has, for. B. glycine.

General methods

The Most of the techniques used to transform cells To construct vectors, to effect hybridization with a probe, To perform site-directed mutagenesis and the like are in the state of Technology widely practiced. Most practitioners are familiar with the standard source materials, describe the specific conditions and processes, familiar (see z. B.

Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (1989)), incorporated herein by reference. For the extra guide however, the following paragraphs will become available shown.

Expression of 2,5-DKG reductase A

The complete functional gene is ligated into a suitable expression vector containing a promoter and a ribosome binding site operable in the host cell into which the coding sequence will be transformed. A number of delivery / control systems and suitable prokaryotic hosts suitable for the present invention are available in the current state of the art. Similar hosts can be used both for cloning and for expression since prokaryotes are generally preferred for cloning DNA sequences. The process of 2-KLG production is most commonly associated with such microbial systems. E. coli K12 strain 294 (ATCC No. 31446) is particularly useful as a cloning host. Other microbial strains used include E. coli strains, such as E. coli strains. BE coli B, E. coli X1776 (ATCC No. 31537) and E. coli DH-1 (ATCC No. 33489). For expression, the aforementioned strains as well as E. coli W3110 (F-, λ-, prototrophic ATCC No. 27325), bacilli such. B. Bacillus subtilus and other Enterobacteriaceae such. Sal monella typhimurium or Serratia marcesans and numerous Pseudomonas species. A particularly preferred group of hosts includes those cultures capable of converting glucose or other commonly available metabolites of 2,5-DKG. Examples of such hosts are generally found within the genera Acetobacter, Gluconobacter, Acetomonas and Erwinia. The taxonomy and nomenclature of these genera are such that different names are sometimes given to the same or similar tribes. For example, Acetobacter cerinus, used in the example below, is also referred to as Gluconobacter cerinus. Examples of particular hosts include, but are not limited to, Erwinia herbicola ATCC No. 21998 (also contemplated as Acetomonas albosesamae in U.S. Patent No. 3,998,697); Acetobacter (Gluconobacter) oxydans subspecies melanozenes, IFO 3292, 3293 ATCC No. 9937; Acetobacter (Gluconobacter) cerinus IFO 3263 IFO 3266; Gluconobacter rubiginous, IFO 3244; Acetobacter fragum ATCC No. 21409; Acetobacter (Acetomonas) suboxydans subspecies industrious ATCC No. 23776.

in the Generally, plasmid expression or cloning vectors will be used or conjugating plasmids, the replication and control sequences which are derived from species that interact with the host cell compatible are used in conjunction with these hosts. The vector usually carries an origin of replication as well as marker genes that are capable are, a phenotypic To provide selection of transformed cells. For example, will E. coli typically transformed using pBR322, a plasmid derived from an E. coli strain (Bolivar et al., Gene, 2: 95-113 (1977)). contains pBR322 Genes for Ampicillin and Tetracycline resistance and thus provides simple means for identification transformed cells. For the use in the expression must be the pBR322 plasmid or a other microbial plasmid also contain promoters, or it must be modified to contain promoters which the microbial organism for the expression of its own proteins can be used. Those Promoters, the most common used in recombinant DNA construction include the β-lactamase (penicillinase) system and lactose promoter systems (Chang et al., Nature, 275: 617-624 (1978); Itakura et al., Science, 198: 1056-1063 (1977); Goeddel et al., Nature, 281: 544-548 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8: 4057-4074 (1980); EP Application No. 0 036 776). While these are the most common Other microbial promoters have been discovered and used. Details regarding their nucleotide sequences were released, which enables the skilled person, those functional in operable To ligate relationship to genes in transformation vectors (Siebenlist et al., Cell, 20: 269-281 (1980)).

By suitable cleavage and ligation may be DNA sequences, the 2,5-DKG reductase A and B encode, included in the aforementioned vectors which, as stated above, were prepared. Any unnecessary or inhibitory sequences destroyed and the prokaryotic enzyme can then be purified; or the intact or broken cells are used directly as catalysts used. Alternatively, the host can be chosen so that he, if Once transformed, it is capable of the entire implementation from glucose or other suitable metabolite to the desired 2-KLG product to effect.

Either the wild-type plasmid DNA ', the mutated plasmid DNA for 2,5-DKG reductase A as well as the mutated plasmid DNA for 2,5-DKG reductase B become a host for transfected the enzyme expression. The recombinant host cells are cultured under conditions involving enzyme expression favor. Generally, selection pressure is provided by the presence an antibiotic. Resistance to the antibiotic is through encodes the vector.

vector construction for the mutagenesis

Anderson et al. described the construction of plasmid ptrpl-35 in U.S. Patent 5,008,193, incorporated herein by reference, which contains the cloned 2,5-DKG reductase A gene under the control of the E. coli trp promoter ( 1 ). A derivative of this plasmid is constructed with a few minor modifications to facilitate the construction and characterization of mutant forms of 2,5-DKG reductase A. These modifications are described below. The final plasmid construct is called pSStac.DKGR.AAA and is in 2 shown.

• A) The structural gene for the 2,5-DKG reductase A is mutated to include three new restriction enzyme sites facilitate further mutagenesis studies. These three sites are "silent," that is, the amino acid sequence of the resulting DKGR-A protein remains unchanged.
• B) The promoter in pSStac.DKGR.AAA is the tac II promoter described by de Boer et al. (Proc Natl Acad Sci., USA, 80: 21-25 (1983)) instead of the trp prompotor found in ptrpl-35. this is a modified version of the trp promoter containing the binding site for the lac repressor, allowing the expression of the gene to be regulated in cells expressing the lac repressor.
• C) The plasmid is further modified to the origin of replication of the single-stranded filamentous To contain phage f1. The use of this DNA sequence in plasmids is well known in the art to be a single-stranded form of the plasmid, for sequencing and mutagenesis.

To generate 2,5-DKG reductases and mutants, two additional plasmids were constructed: ptrpl-35.A and ptrpl-35.B ( 3 ) which express the structural genes for the 2,5-DKG reductase variants A and B, respectively, behind the E. coli trp promoter in a pBR322 -derived vector. The starting point for these plasmid constructs was ptrpl-35 described in U.S. Patent No. 5,008,193.

At the Prepare the genes for DKG Reductase A and B for The expression has been a series of modifications of wild-type coding Generated sequences of these genes. The wild-type DKG reductase A gene plasmid in ptrpl-35 has an EcoRI site immediately upstream of the Start-methionine; a KpnI site became immediately after the stop codon introduced, to enable that the entire structural gene is excised in an EcoRI-KpnI digestion becomes. Similar were EcoRI and KpnI sites immediately upstream and downstream of the wild-type DKG reductase B gene to enable that the B gene is placed in the same vector as the A gene.

The Wild-type DKG reductase A gene was modified to yield new XbaI and Introduce ApaI sites, in common with flanking EcoRI and KpnI sites, causing the gene in three Segments, each about one third of the total length. The XbaI and ApaI sites in the A gene are "silent," that is, they change the amino acid sequence of the encoded protein. The same two XbaI and ApaI sites were introduced into the analogous positions of the DKG reductase B gene. The first of the two sites, XbaI, is silent in the B gene and changes the amino acid sequence not the B gene. It was not possible, however, the second of the two Insert sites (ApaI) into the B sequence without changing the amino acid sequence to change. Sequence variation was therefore introduced to increase the ApaI site Serine 189 of DKG reductase B was added to glycine (the amino acid, the is found in the analogous position of the A gene) during the Creation of the ApaI site mutated.

The Plasmid ptrpl-35 was digested with EcoRI and HindIII to give - 1690 bp great Fragment to produce the structural gene for DKG reductase A and downstream one Contains sequence, which is purified by acrylamide gel electrophoresis and the in EcoRI and HindIII digested M13mp19 vector DNA is ligated. ligation were transformed into cells of E. coli strain JM101 cells; and the appropriate recombinants were prepared by restriction mapping recombinant phage RF preparations identified. The recombinant Phage (M19mp19.EcoR1 / HindIII.DKGRA) was used as a template preparation (single Form) for Mutagenesis reactions in large scale prepared.

Of the Starting point for Plasmids containing the wild type DKGR-B gene is the plasmid pCBR13 as described (Grindley et al., Applied and Environmental Microbiology, 54: 1770-1775 (1988)), incorporated herein by reference. The plasmid pCBR13 was digested with EcoR1 and BamH1 to a 2000 bp fragment which was purified by acrylamide gel electrophoresis and EcoR1 and BamH1 digested M13mp19 was ligated to the recombinant Phage (M13mp19.R1 / BamH1.DKGRB). The recombinant phage (M13mp19.R1 / BamH1.DKGRB) was used as a template preparation (single-stranded form) for mutagenesis reactions in the large scale prepared.

The Mutagenesis reactions were as follows. Oligonucleotide primers were designed to create new restriction sites in the wild-type DKGR-A and Insert B-genes: For DKGR A: XbaI, ApaI and KpnI were introduced; for DKGR B: EcoRI, ApaI, XbaI and KpnI sites have been introduced. (The oligonucleotides used to identify these restriction sites introduce, were the following:

Mutagenesis reactions were by the "two-primer" method as described by Carter, Methods Enzymol., 154: 382 (1987), incorporated herein by reference. Mutagenic oligonucleotides were diluted to 10 OD ₂₆₀ units per ml. A kinase reaction was performed as follows: 2 μl primer, 2 μl 10x kinase buffer, 1 μl 100 mM DTT, 13.5 μl double-distilled and demineralized H ₂ O and 0.5 μl kinase (4 units / μl, New England Biolabs, Beverly, Massachusetts) for 30 minutes at 37 ° C. The kinase was then heat-inactivated by incubation at 70 ° C for 15 minutes and the reaction was adjusted to 5 μM primer concentration. Annealing reactions were set up containing 5 μL of each appropriate primer at a concentration of 5 μM, 5 μL of similar kinase-digested "upstream" primer (a 18-mer sequencing-type complement complementary just upstream of the M13 polylinker cloning site (5 '-TTC CCA GTC ACG ACG TTG-3' (SEQ ID NO: 11), 3μg template, 2.5μl 10x RB buffer in a final volume of 25μl and duplicate strands were heated to 75 ° C in a heating block 3 minutes, it was allowed to cool to 25 ° C on the laboratory bench, extensions were made by the addition of 2 μl of 2.0 mM dATP, dCTP, dGTP and dTTP plus 1 μl of ligase (6 units / ml). μl, New England Biolabs, Beverly, Massachusetts), 1 μl of Klenow fragment of E. coli DNA polymerase (5 units / μl, large fragment of DNA polymerase I, New England Biolabs, Beverly, Massachusetts), 5 μl 5x ligase buffer, 2 ul of 10 mM rATP, and H ₂ O to a total volume of 50 ul. The ET ngerung was carried out at 25 ° C for 4 hours. 5 μl of the extension reaction was transformed into CaCl ₂ -competent MutL-E. coli cells. Single plaques were placed in a 96-well microtiter plate, grown at 37 ° C, stamped on a lawn of cells from E. coli strain JM101 and grown again at 37 ° C. Multiple slices of nitrocellulose filters were taken from each plate and probed with ³² P radiolabelled mutagenic oligonucleotides. The conditions were as follows: hybridization for one hour at 37 ° C, washing with 6xSSC at 37 ° C, then with TMACI wash solution (3M tetramethylammonium chloride, 50mM Tris, pH 8.0, 2mM EDTA and 0.1% SDS at 65 and 68 ° C). Single putative recombinants that hybridized to all of the appropriate olibonucleotides were prepared in a single-stranded form and sequenced to confirm that all proper sites were present and that no secondary mutations had been introduced. The mutated phage identified as a result were named:

The mutant genes were subcloned from the phage back into the ptrpl-35 vector for expression. The template of M13mp19.R1 / HindIII.DKGR.AAA was used with the Klenow fragment of E. coli DNA polymerase (5 units / μl, large fragment of DNA polymerase I, New England Biolabs, Beverly, Massachusetts) all four dNTPs were "filled in" to produce a double-stranded form in the following reaction: 25 μl template, 5 μl 10x nick dns re-synthesis buffer, 3 μl 10 mM dATP, dCTP, dGTP, dTTP, 10 μl M13 complementary "Upstream" primer (5'-TTC CCA GTC ACG ACG TTG-3 ') in a total volume of 52 μl. The reactions were slowly subjected to double stranding in a heating block as before, initiated by the addition of 1 μl of E. coli DNA polymerase Klenow fragment (5 units / μl, large fragment of DNA polymerase I, New England Biolabs, Beverly , Massachusetts) and extended for 30 minutes at 25 ° C. The reaction mixture was then digested with EcoRI and HindIII and the resulting 1690 bp fragment was purified and subcloned into ptrpl-35 which had been digested with EcoRI and HindIII to produce the plasmid ptrpl-35.DKGR.A. For the DKGR B construct, the template M13mp 19.EcoRI / BamH1.DKGR.BBB was similarly filled in, digested with EcoRI and KpnI, and this - 843 bp fragment was purified by acrylamide gel electrophoresis. This fragment was then in EcoRI / KpnI digested cloned ptrpl-35.A (replacing the mutated DKGR A gene but retaining the downstream DKGR A sequences from KpnI to HindIII). This plasmid is referred to as ptrpl-35.DKGR.B: S189G.

The DKGR B expression construct ptrpl-35.DKGR.B: S189G was used as a Starting point used to construct a plasmid containing wild-type DKGR B, with the appropriate codon for serine at position 189. This was carried out as follows: ptrpl-35.B: S189G was digested with NcoI and XhoI to approximately the inner ⁓2 / 3 (⁓700 bp) of the coding sequence, these being Region the introduced XbaI and ApaI sites as well as the mutation of serine to glycine at the amino acid 189. This region has been replaced by the wild-type gene sequence of NcoI to XhoI from pCBR13. The final construct is designated ptrpl-35.DKGR.B.

To generate 2.5-DKG reductase A and B protein for characterization, plasmids ptrpl-35.A and ptrpl-35.B were co-incubated with pBR322 control plasmid into E. coli strain HB101 by CaCl ₂ Procedure and selected on LB altar plates containing ampicillin and tetracycline. Five ml cultures were grown to saturation overnight in LB plus ampicillin plus tetracycline at 37 ° C with shaking. Cells were recovered by centrifugation and aliquots were analyzed by SDS-PAGE gel electrophoresis. No new bands were observed either in the molecular weight range of 30,000 expected for 2,5-DKG reductase in these cell lysates, nor in similar experiments with E. coli strain MM294. Cell lysates were assayed for 2,5-DKG reductase activity and no activity was observed in these lysates over the pBR322 lysate background.

If similar to these plasmids introduced into Acetobacter cerinus strain (IFO 3263), grown at 28 ° C and were controlled for expression by SDS-PAGE electrophoresis, were emerging new bands in the range of 30,000 daltons observed in the ptrpl-35.A and ptrpl-35.B lysates. Investigations of Acetobacter cerinus cell lysates showed 2.5-DKG reducing activity also an increased activity over the pBR322 background.

Site-directed mutagenesis

The DNA sequence encoding 2,5-DKG reductase A or 2,5-DKG reductase B, is subjected to site-directed mutagenesis to replace nucleotides, the selected one amino acids encode at the predetermined positions in the sequence.

The preferred procedure for Site-directed mutagenesis where only a single base pair is altered must be done by cloning the DNA sequence containing the wild-type enzyme into recombinant plasmid containing an origin of replication from a single-stranded one Bacteriophages. Then a suitable primer is used to To transform nucleotide at an identified position. A synthetic one Oligonucleotide primer which, except in the areas of limited missing matches, complementary is to the desired Sequence, is used as a primer in the synthesis of a strand, the complementary is to the single-stranded Sequence of wild-type 2,5-DKG reductase A or 2,5-DKG reductase B in the plasmid vector. The resulting double-stranded DNA is transformed into a host bacterium. Contours of the transformed Bacteria are plated on agar plates, causing colony formation made possible by single cells which contains the plasmid. Theoretically, 50% of the colonies become consist of plasmid containing the mutated form; 50% will be the starting sequence to have. The colonies are labeled with radiolabeled synthetic primer hybridized under stringent conditions, the hybridization only with the mutant plasmid allow that a perfect match will form with the probe. Hybridizing colonies then become picked and cultured and the mutated plasmid DNA is recovered.

subsequent Site-directed mutagenesis can be used to additional To change nucleotides in any mutant. Alternatively, mutants with more than one changed Nucleotide can be constructed using techniques with which practitioners are familiar with, such as B. the isolation of restriction fragments and ligating such fragments into an expression vector (see z. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (1989)).

Selection of sites for mutagenesis of mutants for the wild type 2,5 DKG reductase A gene

Crucial in the selection of mutagenesis sites is the prediction of a secondary and tertiary structure of the wild-type enzyme. The predictions of the secondary structure are done in one of the following ways carried out. First, the sequences of 2,5-DKG reductases A and B and those of five other homologous enzymes (prostaglandin F synthase, bovine lens and rat lens aldose reductase, human liver aldehyde reductase, and ρ-crystallin from frog-eye lenses) are arrayed to a number to uncover preserved remains. Second, the sequences undergo a series of structural prediction algorithms (Chou and Fasman, Adv. Enzymol., 47: 45-148 (1978); Garnier et al., J. Mol. Biol., 120: 97-120 (1978); Biol., 203: 221-232 (1988); Karphus and Schulz, Naturwissenschaften, 72: 212-214 (1985); Eisenberg et al., Proc Natl. Acad Sci. 81: 140-144 (1984); Rose and Roy, Proc Natl Acad Sci., USA, 77: 4643-4647 (1980)), which are well known in the art. These predictions are collected and compared with each other to derive a rough model of the secondary structure of the enzyme as an eight-stranded α / β-barrel ("algorithmic model"). This secondary structure prediction is in agreement with the recently resolved secondary structures of homologous enzymes that fold an eight-stranded α / β-barrel (Rondeau et al., Nature, 355: 469-472 81992); Wilson et al., Science, 257: 81-84 (1992)).

The Barrel structure is composed of two components. The first Component is a core of eight twisted parallel beta strands, the are arranged close to each other to a ton, similar Fassdauben. This barrel structure A second component of eight alpha helices surrounds with the Beta strands are connected by grinding different lengths. These eight-stranded α / β barrel structure is called the triosephosphate isomerase (TIM) -tun after the enzyme, For which this structure was first observed. The folding pattern of the α / β barrel becomes found in 17 enzymes whose crystal structures are known. In indeed, that's about 10% of the known enzyme structures α / β-tons (Faber and Petsko, TIBS, 15: 228-234 81990)). The 17 known α / β-tons enzymes have a common α / β barrel core; this substrate and cofactor specificity comes from the variable loops, the beta strands and Alpha-Helices connects.

A predicted secondary structural model for the 2,5-DKG reductase A based on the algorithmic model (see above) is shown schematically in FIG 4 (SEQ ID NO: 1) where beta strands are represented by arrows and the alpha helices are shown as cylinders. Regions of the polypeptide chain that link the predicted elements of the secondary structure are characterized as being of undefined structure. There are N- and C-terminal extensions of 34 and 17 amino acids, respectively. From some subgroups of the eight loops at the C-terminus of the beta-leaflet (in the direction of the left side of 4 ), as well as the C-terminal "tail" (positions 262 to 278), are believed to comprise the active site of the enzyme, as in other TIM tonsenzymes. Although it is only a crude model, this structure greatly facilitates rational processing of the enzyme by allowing it to focus on those residues found in predicted loops of the active site. It is apparent that additional residues that are close to those in the predicted loops and "tail" may also comprise part of the active site.

The Selection of posts for Mutagenesis is enhanced through another comparative structural analysis. The sequence analysis 2,5-DKG reductase reveals it as a member of a large superfamily monomeric, NADPH-dependent prokaryotic and eukaryotic carbonyl reductases known as the aldo-keto reductases (Carper et al., Exp. Eye Res., 49: 377-388 (1985); Bohren et al., J. Biol. Chem., 264: 9547-9551 (1989)). Members of this Close group biosynthetic enzymes such. B. bovine prostaglandin F synthase, detoxifying enzymes such. B. chlorodeconuctase and aflatoxin b1 reductase, as well as structural proteins without identified enzymatic activity such. B. rho-crystalline from Froschlinsen a.

The structure of human aldose reductase reveals a number of key properties of importance in homology modeling. The aldose reductase α / β barrel is composed of eight beta strands which form the "core" of the barrel, surrounded by eight alpha helices connected to the beta strands by grinding of varying lengths. As in other known TIM tons enzymes, the loops found at the C-terminal ends of the beta strands comprise the active site of the enzyme where substrate and cofactor bind and catalysis occurs. NADPH is bound in an extended conformation at the top of the barrel, with the nicotinamide ring from which the hydride transfer occurs occupying approximately the exact center of the barrel. The orientation of the cofactor nicotinamide ring is what would be expected for an A-class reductase, with the pro-R hydrogen protruding into the substrate binding pocket. There are two additional secondary structure features on the aldose reductase ketone: two additional alpha helices (labeled H1 and H2) that cluster on the loops of those amino acids that connect the beta strand seven and the alpha helix seven, and in the C-terminal " Tail "can be found after the alpha helix eight. The structure of aldose reductase shows from this C-terminal tail that it extends beyond the top of the barrel to a portion of the active Zen to form.

A model of 2,5-DKG reductase variant A was formed based on the coordinates of the aldose reductase: NADPH complex (Wilson et al., Science, 257: 81-84 (1992)) using modeling techniques (Greer, et al. Methods in Enzymology, 202: 239-252 (1991); Bajorath et al., Protein Science, 2: 1798-1810 (1993), both incorporated herein by reference). 5 shows the secondary elements predicted by this model.

Such Information on what amino acids can comprise the active center of an enzyme, from the knowledge of the actual be obtained three-dimensional form of the enzyme in question, such as obtained from X-ray crystallographic or NMR studies. In the case of 2,5-DKG reductase, there are no such equivalents so far Information in the published Literature. Consequently, would an alternative strategy in such a case is the use of the Models for the 2,5-DKG reductase A, as discussed above, for the possible amino acid substitutions or combinations of individual amino acid substitutions to those residues to limit those who were found to be associated with areas of active center are connected.

mutations at certain points in a protein can lead to increased expression lead that protein in bacteria. Many of the other possible ones Point mutations become tightly contiguous in clusters of one to four amino acid substitutions generated. Of the mutants that are stably folded, only those practice an activity which are significantly different from the wild-type enzyme, in the 21-25 range, the 46-52 range, the 164-170 loop, the 188-220 loop, the 230-235 loop and the C-terminal "tail" (262-278). This is an extra Confirmation, that these loop and tail regions are the active center of the Enzyme include.

each Number of mutations predicted herein may be in a single Mutant can be combined. Obviously, a certain substitution closes substitution with another amino acid at the same location in one place in the particular mutant.

The The following examples are presented to illustrate the present invention to explain and to assist a specialist in doing the same and to use. The examples are not considered in any way that they otherwise limit the scope of the invention.

EXAMPLE 1

Construction of the plasmid pSStac.DKGR.AAA for mutagenesis

A Aliquots of plasmid ptrpl-35 were digested with EcoRI and HindIII restriction enzymes and the resulting 1690 base pair fragment was digested Purified by agarose gel electrophoresis. This fragment was then ligated into EcoRI and HindIII digested vector M13 mp19. The resulting recombinant phage (referred to as M13 mp19.DKGRA) was used to create a single-stranded template form of the phage for the subsequent Isolate mutagenesis. At the matrix was a mutation with triggered three oligonucleotides, to introduce three new restriction enzyme cleavage sites in the 2,5-DKG reductase A gene. These In this case, passages are all "dumb," even though they are one introduce new restriction cleavage site into the DNA sequence, wherein the amino acid sequence of the protein, for which is encoded because of the degeneracy of the genetic code unchanged remains. The three mutagenic oligonucleotides and the introduced changes are as follows: 1) the oligonucleotide XbaA has the sequence 5'CGCGAAGCTGGCTCTAGATCAGGTCGAC 3 '(SEQ ID NO: 12) and leads an XbaI site at the amino acid position 98; 2) the oligonucleotide ApaA has the sequence 5 'ATCGTGGGGGCCCCTCGGTCAGGGC 3 '(SEQ ID NO: 13) and leads a new ApaI site at amino acid position 188; and 3) the oligonucleotide KpnA has the sequence 5 'GAGGTCGACTGAGGTACCCGAACACCCG 3' (SEQ ID NO: 14) and leads a KpnI site immediately following the stop codon (TGA) after the last amino acid. The mutagenesis reaction and conditions were essentially the same as in Example 2 for described the construction of the mutant Q192R. After the mutagenesis reaction were positive plaques by hybridization to mutagenic oligonucleotides under stringent conditions and the entire coding The region of the 2,5-DKG reductase A fragment was sequenced to to confirm the mutations.

The plasmid pSStac.DKGR.AAA was constructed as a three-way ligation of the following fragments: 1) EcoRI to HendIII from the mutagenized phage M13 mp19.DKGRA as described above, this fragment being the coding gene for 2,5-DKG Reductase A contains; 2) the PstI to EcoRI fragment (850 base pairs) of the plasmid ptac6 (ptac6 is equivalent to the plasmid ptrpl-35 but contains the tac promoter as described in de Boer et al., (Proc Natl Acad Sci., USA, 80: 21-25 (1983)), instead of the trp promoter found in ptrpl-35, and 3) the 4,000 base pair vector fragment from HindIII to PstI from plasmid p690. The p690 plasmid is a derivative of plasmid pBR322 having the RsaI / DraI restriction fragment from the bacteriophage f1 genome (nucleotides 5489-5946) containing the single-stranded DNA replication origin inserted into the PouII site.

The three fragments described above were isolated by agarose gel electrophoresis, purified and ligated in approximately equimolar ratios and used to transform competent E. coli cells. The resulting colonies were analyzed by restriction mapping to identify the correct construct, designated pSStac.DKGR.AAA ( 2 ).

EXAMPLE 2

Site-directed mutagenesis of the 2,5-DKG reductase A gene

A. Preparation of template DNA for the mutagenesis

E. coli cells (strain XL1-Blue, Stratagene Corporation) containing the plasmid pSStac.DKGR.AAA were amplified in LB media (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press A.1 (1989)) Grow until the early days logarithmic phase and with the helper phage VCS-M13 (Stratagene) infected. Infection with a helper phage sets the required factors for the Packaging and secretion of the single-stranded form of the plasmid pSStac.DKGR.AAA ready. The infected cells were shaken overnight Grow to 37 ° C left and next Day, the cells were removed by centrifugation at 10,000 rpm for 10 Minutes in a Sorvall SM24 rotor. The supernatant containing the packaged plasmid contains was saved and the cell pellet discarded. The packaged plasmid was made by adding 1/4 volume of 2.5 M NaCl, 20% PEG (polyethylene glycol) like. After the addition, the mixture was stored at 25 ° C for 20 minutes and then became the precipitate removed by centrifugation.

The precipitate was dissolved in 0.4 ml of TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) and further purified by several successive extractions with an equal volume of 50:50 chloroform: phenol. After each extraction, the aqueous (upper) phase was stored. The DNA was precipitated with 2 volumes of ice-cold ethanol. The precipitate was recovered by centrifugation and dissolved in TE buffer. The concentration of the plasmid was estimated by measuring the optical absorbance at 260 nm using the conversion of 1 OD ₂₆₀ = 40 μg of single-stranded DNA per milliliter. The concentration of the plasmid was adjusted to 1 μg per ml with TE.

B. phosphorylation of oligonucleotide primer

A synthetic oligonucleotide having the sequence 5 'GCCCCTCGGTCGCGGCAAGTACG 3' (SEQ ID NO: 15) was synthesized and phosphorylated as follows: The oligonucleotide was diluted to a concentration of 5.0 OD ₂₆₀ units per ml. Then, 2.5 oligonucleotide with 3 μl 10x kinase buffer (1 M Tris, pH 8.0, 100 mM MgCl ₂ , 70 mM dithiothreitol, 10 mM ATP), 25 μl water and 2 units T4 polynucleotide kinase (4 units / μl, New England Biolabs, Beverly, Massachusetts). The mixture was incubated at 37 ° C for 15 minutes, then the kinase enzyme was inactivated by heating to 70 ° C for 10 minutes.

C. Mutagenesis reaction

6 μl of with Kinase-treated primer were mixed with 1 μg template DNA and 2.5 μl 10x RB buffer (70 mM Tris, pH 7.5, 50 mM mercaptoethanol, 550 mM NaCl and 1 mM EDTA) in one Total volume of 10.5 μl combined. The primer was formed with the template to double strand by heating the mixture to 65 ° C for 5 minutes, and then slowly cooling down to 25 ° C over a Period of 30 minutes.

To the annealing mixture was added 1.5 μl of 10x RB buffer, 1 μl of 10 mM ATP, 1 μl of 10 mM DTT (dithiothreitol) and 1 μl of T4 DNA ligase (6 Whites / μl, New England Biolabs, Beverly , Massachusetts). After 10 minutes, 1 μl of 1 M MgCl ₂ , 1 μl of 5 mM dNTPs (an equimolar mixture of dATP, dCTP, dGTP and dTTP) and 0.5 μl Klenow (5 units / μl, large fragment of DNA polymerase I, New England Biolabs, Beverly, Massachusetts) and the mixture was incubated at 15 ° C overnight.

The following day, the frozen competent E. coli MutL cells with an aliquot of the Reak transformed mixture and plated on agar plates containing antibiotic selection (12.5 ug / ml tetracycline, 50 ug / ml ampicillin). Colonies carrying mutated plasmids were initially identified by hybridization with the mutagenic starting oligonucleotide under stringent conditions (Wood et al., Proc Natl. Acad Sci., USA, 82: 1585-1588 (1988)). Mutant plasmids were then prepared in a single-stranded form as in Section A and confirmed by direct DNA sequencing of the plasmid (United States Biochemical Corporation, Sequenase Sequencing Kit). The resulting mutated Q192R-2,5-DKG reductase A as shown in Example 5 had improved catalytic activity as compared to the wild-type 2,5 DKG reductase A.

EXAMPLE 3

Expression of wild-type 2,5-DKG reductase A in Acetobacter cerinus

Plasmid DNA was introduced into Acetobacter cerinus (ATCC No. 39140) by electroporation as described (Wirth et al., Mol. Gen. Genet., 216 (1): 175-177 (1989)) using a Genepulser apparatus (Biorad Corporation). The cells were grown to mid-log phase (OD ₅₅₀ ⁓0,2-0,8) in 100 ml LB medium and recovered by centrifugation at 5,000 rpm in a Sorvall SS-34 rotor for 5 minutes at 4 ° C. The cells were resuspended in half a volume of ice-cold electroporation buffer (300 mM sucrose, 7 mM sodium phosphate buffer, pH 7.0 and 1 mM MgCl ₂ ), pelleted again by centrifugation and then resuspended in 1/20 volume of electroporation buffer and stored on ice until use ,

Plasmid DNA (0.1 to 1.0 μg) became a 0.4 cm electroporation cuvette (Biorad Corporation) added which contained 0.8 ml of the prepared Acetobacter cells. The Cells and DNA were mixed in the cuvette and placed on ice for 10 Chilled minutes before electroporation. The cells and the DNA was a single pulse of 2500 mV using a 25μF capacitor setting and immediately diluted to 3.0 ml with fresh LB media. The diluted Cells was then allowed with shaking 30 ° C for 2 hours to recover. Aliquots (10-100 μl) of the transformed Cells were seeded on selection media (LB altar plates containing 50 μg / ml Ampicillin and 12.5 μg / ml Tetracycline) and the plates were plated overnight at 30 ° C let grow.

EXAMPLE 4

Purification of the mutant Q192R and wild type 2,5 DKG reductase A

Single colonies of transformed Acetobacter cerinus cells were dissolved in 200 ml 2 X YT media (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)) containing antibiotics (12.5 μg / ml tetracycline and 50 μg / ml Ampicillin) at 30 ° C overnight let grow. The cells were recovered by centrifugation won (15 minutes at 8000 rpm in a Sorvall GS3 rotor) and stored frozen. The cells were then transferred to 1/5 volume lysis buffer (50 mM Tris, pH 8.0, 50 mM EDTA, 0.1% Tween, 2 mg / ml lysozyme) and for Lysed for 2 hours on ice. The lysed cells were in turn centrifuged as before and the supernatant, which contained the crude cell extract was stored.

The 2,5-DKG reductase A protein was purified from the crude cell extract by chromatography purified on DEAE cellulose. DEAE cellulose (Whatman DE-52, Brand) was pre-equilibrated with 25 mM Tris, pH 7.0. A total volume of 5.0 ml of the gel was poured into a disposable plastic chromatography column, on which the crude cell extract was applied. After the whole Extract to the column became the pillar with two column volumes 25 mM Tris, pH 7.0, then with a volume of 25 mM Tris, pH 7.0 containing 0.3 M NaCl, followed by washing containing 2,5-DKG reductase A protein with 25 mM Tris, pH 7.0 0.6 M NaCl, eluted. The preparations were based on the protein concentration by the dicinchonic acid method (Methods in Enzymology, 182: 60-62 (1990)) and checked for their purity by SDS-polyacrylamide gel electrophoresis.

EXAMPLE 5

Kinetic characterization wild-type and mutant Q192R-2,5-DKG reductase A

The preparations of the wild-type and mutant Q192R-2,5-DKG reductase A enzymes were kinetically characterized as their ability to reduce the substrate 2,5-DKG to 2-KLG. The studies were performed in 1 ml total volume of 50 mM Tris, pH 7.0, containing 0.2 mM NADPH, a constant enzyme amount (15-20 μg) and substrate amounts varying from 2 to 14 mM. The studies were performed at 25 ° C and the rate of substrate reduction was measured spectrophotometrically by measuring the loss of absorbance at 340 nm wavelength (which is characteristic of the oxidation of the cofactor NADPH to NADP +).

The data were analyzed according to the well-known Michaelis equation to determine the kinetic parameters Vmax and Km using the Enzfit software package (Biosoft, Cambridge, UK) on an Epson desktop computer. The wild-type 2,5-DKG reductase A had an apparent Vmax for the substrate 2,5-DKG of 7.8 μM per minute per milligram of protein, while the Q192R mutant had an apparent Vmax of 14.0, a 1 , 8-fold increase. The Km or Michaelis constant of the wild-type enzyme appeared to be 28 mM, whereas the Km value of the Q192R mutant appeared to be 21 mM for this substrate. This resulted in an apparent specificity constant (kcat / Km) of 140 M ^-1 s ^-1 for the wild-type enzyme and a specificity constant of 335 M ^-1 s ^-1 for the Q192R mutant, a 2.4-fold increase.

EXAMPLE 6

Homology model of 2,5-DKG reductase A

One Model of 2,5-DKG reductase variant A was formed based on the coordinates of aldose reductase: NADPH complex (Wilson et al., Science, 257: 81-84 (1992)) using modeling methods (Greer, Methods in Enzymology, 202: 239-252 (1991), incorporated herein by reference; Bajorath et al. Protein Science, 2: 1798-1810 (1993); incorporated herein by reference) in which "structurally conserved Areas "(in general Areas with secondary structure properties such as Alpha helix and beta sheet or areas with extensive sequence identity) and are kept constant, to which later "loops" of variable amino acids are added. The conformation of these loops is determined either by searching the Conformation modeled using the crystal structure databases or by chance Konformationsbildungsalgorithmen.

6 shows the protein sequence arrangement of 2,5-DKG reductases A and B with human aldose reductase; Outlined regions show secondary structural properties of the aldose reductase crystal structure (Bruce et al., Biochem J., 299: 805-811 (1994)). In this modeling, the main changes were: replacement of the long loop connecting the beta strand four and the alpha helix four or beta strand seven and the helix H1 by shorter loops and modeling a new tail conformation around the shorter tail the 2,5-DKG reductase A account. The remaining structure was kept constant. Similar looking structures for these loops have been selected from a number of possibilities, generated by a random conformational program. The model was used to target a number of residues in the predicted active site of 2,5-DKG reductase for mutagenesis, and is also used in the following sections to estimate the approximate locations of these mutants in 2.5 Explain -DKG reductase barrel structure.

EXAMPLE 7

Construction of the F22Y mutant the 2,5-DKG reductase A

The Homology model was used to revise structural differences to determine the substrate binding pocket around which the base for differences in the observed substrate conversion rate of 2,5-DKG reductase A and DKG reductase B could be. In particular, the homology model was used to add amino acids locate for a replacement associated with the substrate binding pocket of 2,5-DKG reductase A, which was less hydrophilic than the counter amino acid in 2,5-DKG reductase B.

The amino acid 22 appears to be part of the active site of the substrate specificity pocket in both the aldose reductase structure and the 2,5-DKG reductase A homology model to build. A phenylalanine becomes in the 2,5-DKG reductase A enzyme found while a tyrosine this position in the sequence of 2,5-DKG reductase B occupied. The additional Hydroxyl moiety of tyrosine, compared to phenylalanine, could be active center region of the 2,5-DKG reductase B enzyme H-binding ability to lend.

The Construction of these two mutants, F22Y and Y23F, is as follows: four oligonucleotides, two for the Mutagenesis and two for the probing was designed as follows:

The Oligonucleotides with the appendix ".m" were labeled with kinase treated and used to generate templates for the wild-type 2,5 DKG reductase A and B genes to mutate with the Amersham kit (templates are EcoRI-KpnI fragments the genes for the DKG reductases A and B in M13mp19). The steps in the mutagenesis reactions, the isolation and characterization of the mutants were essentially the same as stated for the Construction of the Q192R mutant, with the exception of the oligonucleotides with the appendix ".p", which uses were to isolate the mutants. The resulting F22Y mutant of 2,5-DKG reductase A has a tyrosine at position 22 and the resulting Y23F mutant 2,5-DKG reductase B has a phenylalanine at position 23.

EXAMPLE 8

Kinetic characterization the F22Y mutant 2,5-DKG reductase A and the Y23F mutant 2,5-DKG reductase B

The kinetic characterization of the 2,5-DKG reductase A mutant F22Y and the 2,5-DKG reductase B mutant Y23F was carried out in substantially the same manner as in Example 5, except that, To determine the kinetic parameters for the NADPH-dependent reduction of 2,5-DKG by the 2,5-DKG reductases, a series of reactions with constant saturation concentration of NADPH (200 μM) and varying concentrations of substrate from 0 to 30 mM were performed. The 2,5-DKG reductase A mutant F22Y shows a significantly and reproducibly increased activity in comparison with the wild-type 2,5-DKG reductase A ( 7 ). The activity of 2,5-DKG reductase B mutant Y23F is lower than that of wild-type 2,5-DKG reductase B (FIG. 7 ). The 2,5-DKG reductase A mutant F22Y could also show increased resistance to substrate inhibition as compared to the wild type 2,5 DKG reductase B enzyme.

EXAMPLE 9

Construction of the I49N mutant the 2,5-DKG reductase A and the N50A mutant 2,5-DKG reductase B

position 49 was for selected the mutagenesis, based on the proximity this location to the substrate binding site in the homology model and a predicted hydrophobicity difference of that position in the 2,5-DKG reductase A and B enzymes: the 2,5-DKG reductase A has isoleucine at position 49, whereas 2,5-DKG reductase B has asparagine at position 50. The position 49 will open a loop of amino acids found that joins the beta strand 2 and the alpha helix 2. Two mutants were constructed to reduce the effect of side chain mutation to investigate: I49N, the 2,5-DKG reductase A mutant, and the 2,5-DKG reductase B mutant N50A. The construction of these mutants was as follows: the oligonucleotide sequences were as follows: A: I49N.m = 5'-C

The Steps in the mutagenesis reactions, isolation and characterization the mutants were essentially the same as explained for construction the F22Y mutant. The resulting I49N mutant has 2,5-DKG reductase A. an asparagine at position 49 and the resulting N50A mutant 2,5-DKG reductase B has an alanine at position 50.

EXAMPLE 10

Kinetic characterization the I49N mutant 2,5-DKG reductase A and the N50A mutant 2,5-DKG reductase B

The kinetic characterization of the 2,5-DKG reductase A mutant I49N and the 2,5-DKG reductase B mutant N50A was performed in substantially the same manner as in Example 5, except that To determine the kinetic parameters for the NADPH-dependent reduction of 2,5-DKG by 2,5-DKG reductases, a series of reactions with constant saturation concentration of NADPH (200 μM) and varying substrate concentrations from 0 to 30 mM were performed. The 2,5-DKG reductase A mutant I49N did not produce any detectable levels of recombinant protein in the host cell, possibly due to structural instability. The 2,5-DKG reductase B mutant N50A expressed normally. Kinetic results for the 2,5-DKG reductase B mutant N50A are in 8th shown. The 2,5-DKG reductase B mutant N50A exerted no substrate inhibition up to substrate concentrations higher than 15 mM.

The enzyme activity of the wild-type 2,5-DKG reductase B weakens after the addition of only 5 mM 2,5-DKG ( 8th ).

EXAMPLE 11

Construction of 2,5-DKG reductase A mutants D278A, V277A, E276A, D275A, P274A, H273A, A272G, S271A, V270A, R269A, S267A and D265A of 2,5-DKG Reductase A

The Technique of "ala-scanning" was used to localize residues in the C-terminus of 2,5-DKG reductase A (Cunningham and Wells, Science, 204: 1081 (1989)), incorporated herein by reference Reference. A total of 11 ala-scan mutants were constructed: D278A, V277A, E276A, D275A, P274A, H273A, S271A, V270A, R269A, S267A and D265A, based on the prediction that by these mutants covered area was part of the active site of the enzyme. In addition was the following non-ala-scan mutant generates: the 2,5-DKG reductase A mutant A272G was created by replacing the alanine at position 272 with a Gycin in that position. The 2,5-DKG reductase mutants were constructed using the following oligonucleotides:

The Steps in the mutagenesis reactions, isolation and characterization the mutants were essentially the same as for the construction the Q192R mutant.

EXAMPLE 12

Kinetic characterization the 2,5-DKG reductase mutants D278A, V277A, E276A, D275A, P274A, H273A, A272G, S271A, V270A, R269A, S267A and D265A of 2,5-DKG reductase A

A crude kinetic characterization of the 2,5-DKG reductase A mutants D278A, V277A, E276A, D275A, P274A, H273A, A272G, S271A, V270A, R269A, S267A and D265A was performed. One of these mutants, A272G, resulted in increased activity. The 2,5-DKG reductase A mutant A272G was characterized in substantially the same manner as in Example 5, except that to give kinetic parameters for the NADPH-dependent reduction of 2,5-DKG by the To determine 2,5-DKG reductases, a series of reactions were performed with constant saturation concentrations of NADPH (200 μM) and varying concentrations of substrate from 0 to 30 mM. The mutant 2,5-DKG reductase A mutant A272G showed significant and reproducible activity against the wild-type A enzyme at all substrate concentrations, with only minor evidence of substrate inhibition in the region examined (see 9 ). The mutant exerted an apparent Vmax of 21.44 +/- 4.10 sec ^-1 and an apparent Km of 42.61 +/- 12.13 mM.

EXAMPLE 13

Construction of 2,5-DKG reductase A double mutants F22Y / Q192R, Q192R / A272G and F22Y / A272G

Three 2.5 DKG reductase A double mutants were constructed: F22Y / Q192R, Q192R / A272G and F22Y / A272G. The constructs were as follows:

For the Q192R / A272G double mutant the plasmid ptrpl-35.A: Q192R was digested with EcoRI and BamHI, to generate a fragment of 787 bp long, the Q192R mutation contains. The plasmid ptrpl-35.A: A272G was digested with BamHI and ClaI to to generate a 708 bp long fragment containing the A272G mutant. The Both mutants were in a three-way ligation with the EcoRI and ClaI digested vector ptrpl-35.A combined to the double mutant ptrpl-35.A: Q192R / A272G to create. The mutants were verified by restriction digestions, to ensure that both expected fragments are in place were. The resulting Q192R / A272G mutant 2,5-DKG reductase A has an arginine at position 192 and a glycine at position 272nd

For the 2,5-DKG reductase double mutant F22Y / A272G became a F22Y-containing 600 base pair fragment by EcoRI and Apal digestion of plasmid ptrpl-35.A: F22Y. A fragment 0300 bp) carrying the mutation A272G was detected by ApaI and KpnI digestions of the plasmid prtpl-35.A: A272G prepared. The mutant was then in a three-way ligation with EcoRI-KpnI-digested ptrpl-35.A combined to give the plasmid ptrpl-35.A: F22Y / A272G result. The resulting F22Y / A272G mutant 2,5-DKG reductase A has a tyrosine at position 22 and a glycine at position 272nd

The Mutant 2,5-DKG reductase A F22Y / Q192R was replaced by a similar one Strategy, the oligonucleotide containing the Q192R mutation ruled, but removed the ApaI site, allowing the construct to pass through the XhoI site of the DKG reductase A gene was made. The plasmid ptrpl-35.A: F22Y was digested with EcoRI and XhoI, yielding a 435-bp fragment which contains the F22Y mutation. In a second digestion ptrpl-35.A: Q192R was digested with XhoI and KpnI to give a 400 bp to give a long fragment containing the Q192R mutation. These Both fragments were in a three-way ligation with EcoRI and KpnI-digested ptrpl-35.A combined to form the plasmid ptrpl-35.A: F22Y / Q192R to surrender. The resulting F22Y / Q192R mutant has the 2,5-DKG reductase A. a tyrosine at position 22 and an arginine at position 192.

All three double mutants were identified by restriction mapping and direct Sequencing confirms to Make sure the two correct mutations are in place and Place were.

EXAMPLE 14

Kinetic characterization the 2,5-DKG reductase A double mutant F22Y / Q192R, Q192R / A272G and F22Y / A272G

The kinetic characterization of the 2,5-DKG reductase A double mutants F22Y / Q192R, Q192R / A272G and F22Y / A272G were carried out in substantially the same manner as in Example 5, except that to assess the kinetic parameters for the NADPH-dependent reduction of 2,5-DKG by 2.5-DKG To determine reductases, a series of reactions with constant saturation concentration of NADPH (200 uM) and varying substrate concentrations from 0 to 30 mM were performed. The substrate kinetics for the double mutants are in 10 shown. Double mutants containing Q192R did not result in increased activity. The catalytic activity of the 2,5-DKG reductase A mutant F22Y / Q192R is similar to that of the two parent mutants. The catalytic activity of the 2,5-DKG reductase A mutant Q192R / A272G is lower than that of the wild-type DKG reductase A. The 2,5-DKG reductase A mutant F22Y / A272G double mutant has a clear Additivity or even synergy with their two parent enzymes and exceeds the activity of DKG reductase B at substrate concentrations higher than 17.5 mM. This double mutant also shows a substrate inhibition effect.

EXAMPLE 15

Cofactor Kinetic Characterization the 2,5-DKG reductase A mutants F22Y, Q192R, A272G and F22Y / A272G

The cofactor affinity of 2,5-DKG reductase A F22Y, Q192R, A272G and F22Y / A272G was determined by analysis of a series of reactions with constant substrate concentration and varying concentrations of NADPH. Each series of reactions consisted of 50 mM bis-Tris, pH 6.8, 10 mM 2,5-DKG, from 2.5 to 200 μM NADPH and enzyme in a total volume of 1.0 mM. Initial rate data for the reactions were fitted to the Michaelis-Menten equation by non-linear regression analysis as previously described to determine KM, NADPH. The results are in 11 shown. Deviations from the Michaelis constant for NADPH between the mutants are not significant; values are all within +/- 30 of the KM, NADPH of the wild-type enzyme, ranging between a high level of 8.19 μM for 2,5-DKG reductase A F22Y / A272G and a low level of 4.92 μM for 2,5-DKG reductase A A272G.

EXAMPLE 16

Mutated thermal stability of 2,5-DKG reductase A mutants F22Y, Q192R, A272G and F22Y / A272G

Thermal instability can be a critical property of an enzyme that can limit its usefulness in an industrial process. The 2,5-DKG reductase A mutants F22Y, Q192R, A272G and F22Y / A272G with enhanced catalytic activity were subjected to circular dichroism analysis to determine what effect the mutations might have on thermal stability. Protein samples of 2,5-DKG reductase A and B and 2,5-DKG reductase A mutants F22Y, Q192R, A272G and F22Y / A272G were concentrated over Amicon YM-10 membranes desalted in 10 mM phosphate buffer , pH 7.0, using a pre-packed G25 column (Pharmacia PD-10) and adjusted to a final concentration of 200 μg / ml for circular dichroism analysis. Samples were measured in an Aviv Model 60DS circular dichroism spectrophotometer in a 1.0mm cuvette. The measurements were corrected against the buffer background. Raw ellipticity data (degrees) were converted to molar ellipticity values (degrees M ^-1 cm ^-1 ) by the relationship: molar ellipticity = (100) (ellipticity) / C) (1), where C is the molar concentration of the sample and 1 is the path length in centimeters. The proteins showed minima at ⁓220 nm, which is indicative of alpha-helix content. Thermal denaturation was determined by monitoring the loss of ellipticity at 220 nm as a function of temperature. Tm values from the midpoint of the thermal denaturation curves are in 12 shown.

SEQUENCE LISTING

(1) GENERAL INFORMATION

• (i) REGISTERS: POWERS, DAVID B. ANDERSON, STEPHEN
• (ii) TITLE OF THE INVENTION: IMPROVED METHOD OF MANUFACTURING OF VITAMIN C
• (iii) NUMBER OF SEQUENCES: 35
• (iv) CORRESPONDENZADDRESS: (A) ADDRESS: HOWREY & SIMON (B) ROAD: 1299 PENNSYLVANIA AVENUE, N.W. (C) CITY: WASHINGTON (D) STATE: DC (E) COUNTRY: US (F) Post code: 20004
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• (vi) DATA OF THE PRESENT PATENT APPLICATION: (A) REGISTRATION NUMBER: US 08 / 585,595 (B) REGISTRATION DATE: 16 January 1996 (C) CLASSIFICATION:
• (vii) PREVIOUS DATA PATENTS: (A) REGISTRATION NUMBER: US 08 / 584,019 (B) REGISTRATION DATE: January 11, 1996
• (viii) APPLICANT / REPRESENTATIVE INFORMATION: (A) NAME: AUERBACH, JEFFREY L (B) REGISTRATION NUMBER: 32680 (C) REFERENCE / FILE NUMBER: 6137-0014 CIP
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Claims (15)

Mutant form of 2,5-DKG reductase A (2,5-diketogluconic acid reductase A) with improved ability 2,5-DKG to 2-KLG (2-keto-L-gluconic acid) and with an amino acid substitution in position 22 as defined in SEQ ID NO: 1. Mutant according to claim 1 with a tyrosine in position 22. Mutant according to claim 1 with a serine, threonine, histidine, glutamine, asparagine or Tryptophan in this position 22. Mutant according to claim 1 or 2 with an amino acid substitution at position 272 as defined in SEQ ID NO: 1. Mutant according to claim 4 with a glycine in position 272. Mutant form of 2,5-DKG reductase A after one of claims 1, 2, 4 and 5 with reduced substrate inhibition. Mutant form of 2,5-DKG reductase A according to one the claims 1, 2, 4 and 5 with improved temperature stability. Mutant form of 2,5-DKG reductase A after one of claims 1, 2, 4 and 5 with increased Change number of the substrate by the enzyme or an increased affinity for the Substrate. A DNA construct comprising a structural gene containing at least one mutated codon, said gene being a mutant one of the preceding claims coded. Host cell transformed with an expression vector, the one DNA construct according to the claim 9 contains. Host cell according to claim 10, which is a bacterium. The host cell of claim 11, wherein the bacterium from the genus Erwinia, Gluconobacter or Acetobacter. The host cell of claim 12, wherein the bacterium Acetobater cerinus (IFO 3263) is. The host cell of claim 10, wherein the expression vector is a plasmid. The host cell of claim 10, wherein the expression vector ptrpl-35.A is as in 3 with the mutations F22Y / A272G or F22Y / Q192R.